Device and method for measuring prothrombin time and hematocrit by analyzing change in reactance in a sample

ABSTRACT

Devices and methods for measuring prothrombin time (PT) and hematocrit (HCT) by analyzing the change in reactance in a sample are presented. A diagnostic device for measuring HCT and PT of a fluid includes a relative electrode-type sensor device and a blood test card assembly including one or more pairs of electrodes, wherein alternating current (AC) provided by the sensor device is used to measure and calculate HCT and PT of blood test using the reactance analysis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority fromU.S. Provisional Application No. 61/353,137, filed on Jun. 9, 2010, theentire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to biochemical diagnostic devices, andmore particularly, to devices and methods for determining hematocrit(HCT) and prothrombin time (PT) by analyzing a change in reactance in asample.

BACKGROUND OF THE INVENTION

Two pathways or coagulation cascades, known as the intrinsic andextrinsic pathways, lead to the formation of a clot in blood. When ahuman body is injured, the extrinsic pathway is first triggered tocontrol the body's blood coagulation. In addition to a blood sample, thecoagulation reaction needs some additional tissue factors. The inactivefactor X is catalyzed into factor Xa. The prothrombin (factor II) can betransformed from the factor Xa to the thrombin (factor IIa) by theeffects of factor Va, acidic phospholipids and calcium ions. Thethrombin then transforms fibrinogen into fibrin, enhancing the plateletof the endothelial cells gathered at the injury. The thrombin can alsoenhance the role of factor XIII, linking each fibrous protein moleculeto a stable fibrin. Therefore, inspecting the prothrombin time not onlyallows determining whether the function of external activation factorsof the coagulation system are normal, but also allows assessing andmonitoring oral anticoagulants treatment, liver function, vitamin Kdeficiency, coagulation factor deficiency, and disseminatedintravascular coagulation (DIC) syndrome.

Conventional inspection methods for measuring the prothrombin timeanalyze the condensation phenomenon of transforming the serum solubleprotein into an insoluble protein during blood coagulation. Theseinspection methods can be realized by detecting optical characteristics,such as changes in color, reflection, refraction, luminescence andfluorescence. Such inspection methods, however, require a substantialnumber of blood test samples and high purity reagents and aretime-consuming, as disclosed in U.S. Pat. No. 5,418,141, the entirety ofwhich is hereby incorporated by reference. Moreover, these inspectionmethods require long detection times and a significant amount ofsupplies, resulting in inconvenience and higher costs.

Other conventional inspection methods for measurement of the prothrombintime use electrochemical inspection methods. For example, U.S. Pat. No.3,699,437, the entirety of which is hereby incorporated by reference,discloses observing the comparative decline rate of resistance from theinitial to the lowest point. The calculated result is served as a basisfor determining coagulation time in which the impedance measurement isrelated to the mechanism of blood coagulation. Further, U.S. Pat. Nos.6,060,323; 6,338,821; 6,066,504; 6,673,622; and 6,046,051, the entiretyof each of which is hereby incorporated by reference, discloseelectronic sensor devices and a test card assembly for the measurementof the coagulation time of a blood sample. The test card assembly isdesigned with a single electrode or a plurality of electrodes accordingto the measurement demands of the device. The sample is contacted withthe electrodes, which measure the change in impedance corresponding tothe change of viscosity of the blood sample as it coagulates. Thistechnique, however, may result in test errors due to the hematocrit andthe electrolyte concentration differences among blood test samples inindividuals. U.S. Pat. No. 7,005,857, the entirety of which is herebyincorporated by reference, discloses a coagulation inspection devicewith automatic collection of blood samples. The coagulation inspectiondevice determines the coagulation time by measuring capacitance orimpedance changes between two electrodes. These technologies may,therefore, improve the simplicity of the detection device, but theycannot achieve the relatively higher precision and accuracy that theabove-discussed optical detection methods may achieve.

Accordingly, a new biosensor device is needed and described herein formeasuring prothrombin time (PT) and hematocrit (HCT), one capable ofoperating with short test times, simple procedures for the user andwhile achieving highly accurate results.

SUMMARY OF THE INVENTION

One aspect of the present invention is to use reactance measurementstaken from a sample to calculate prothrombin time (PT). As describedherein, using reactance measurements as opposed to impedancemeasurements provides a more accurate analysis of the blood'scharacteristics, reduces the chance of a test error, and improvesmeasurement accuracy.

Another aspect of the present invention is to provide a detection systemand a measurement method for determining prothrombin time (PT) andhematocrit (HCT) of a blood sample using a reactance analysis of thesample. In one embodiment, the detection system includes a sensor deviceand a test card assembly. The test card assembly includes one or morepairs of the carbon or precious metal electrodes, set on the same planeor on different planes, respectively. Alternating current (AC) providedby the sensor device is used to measure reactance and calculate the PTand HCT of the blood sample using the reactance analysis describedherein.

Another aspect of the invention is to provide a test card assembly withan improved blood sample and reagent contact area. The test cardassembly according to some embodiments of the present invention utilizesporous materials, such as fiberglass substrate (FR-4), for at least aportion of a substrate of the test card. Since the surface of at least aportion of the substrate, preferably the majority of the surface of thesubstrate that comes in contact with the sample, is porous, e.g., it mayhave a plurality of holes, voids, or cavities thereon, the sample (e.g.,blood) is improvably, uniformly dispersed on the substrate, therebyincreasing the contact area of the blood sample and reagents, andeffectively improving on drawbacks of traditional non-porous materialsused for substrates. According to the present invention, the problemsassociated with having a relatively poor contact between the sample andthe reagent that occur when using non-porous materials for thesubstrate, or a portion thereof, e.g., relatively high blood cohesion,are minimized or eliminated.

According to one aspect of the invention, a diagnostic device formeasuring PT and HCT of a fluid includes: a electrode-type sensordevice; and a blood test card assembly including one or more pairs ofelectrodes, wherein alternating current (AC) provided by the sensordevice is used to measure and calculate prothrombin time and HCT of theblood test using the reactance analysis described herein.

In one embodiment, the sensor device includes: a test card receivingunit for accommodating the test card assembly; a temperature maintainingunit for controlling and maintaining temperature of the test cardreceiving unit at a constant temperature; an AC generation unit forproviding an alternating current with predetermined frequency andvoltage to the test card assembly; a signal receiving unit to receive aresponse signal from the test card assembly; a microprocessor forcalculating the response signal and rendering results of the HCT and theprothrombin time; and a display unit for displaying inspected results ofthe HCT and the prothrombin time from the microprocessor.

According to another aspect of the invention, a diagnostic device formeasuring HCT and/or PT of a sample includes: an electrode-type sensordevice; a test card assembly including one or more pairs of electrodes;a power source for providing an AC test signal at a constant frequencyto electrodes of the test card assembly; a signal retrieving unitcoupled to the test card assembly; a phase angle calculating unit; amicroprocessor in the sensor device programmed for analyzing a responsesignal from the test card assembly, an output from the phase anglecalculating unit and performing a reactance analysis of a sampleprovided on the test card assembly; and a display for displaying PTand/or HCT of the sample.

According to another aspect of the invention, a method for measuring HCTand/or PT of a sample includes: providing a test card assembly to a testcard receiving unit; controlling and maintaining temperature of the testcard receiving unit at a constant temperature; providing a sample to beinspected to the test card assembly; providing an alternating currentwith predetermined frequency and voltage to the test card assembly by anAC generation unit; receiving a response signal from the test cardassembly and calculating the HCT and/or PT by a microprocessor; andproviding an inspected result to a display unit.

According to an additional aspect of the invention, a method formeasuring HCT and/or PT of a sample includes: providing a test cardassembly to a test card receiving unit; providing a sample to beinspected to the test card assembly; providing an alternating currentwith predetermined frequency and voltage to the test card assembly by anAC generation unit; receiving a response signal from the test cardassembly; performing a reactance analysis of the sample; and determiningthe HCT or the prothrombin time with a microprocessor; and providing aninspected result to a display unit.

According to yet another aspect of the invention, a method for measuringHCT and/or PT of a sample includes: coupling a test card having asubstantially porous surface on the substrate at a sample region of thetest card to a sensor unit programmed to measure HCT and/or PT of ablood sample; providing a blood sample to be analyzed to the test cardsample region; providing an alternating current with a predeterminedfrequency and voltage to the test card; sensing a response signal fromthe sample on the test card; performing a reactance analysis of thesample; determining a phase change related to the capacitance in theblood sample using a microprocessor programmed to determine the phasechange caused due to reactance in the blood sample; and determining theHCT and/or PT of the bloods sample with the microprocessor; andproviding an inspected result to a display unit of the sensor unit.

BRIEF DESCRIPTION OF THE FIGURES AND PICTURES

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated by reference to thefollowing detailed description, when taken in conjunction with theaccompanying pictures, wherein:

FIG. 1 is a schematic view of an exemplary diagnostic device formeasuring HCT and/or PT in accordance with embodiments of the presentinvention;

FIG. 2 illustrates an explosive view of a blood test card in accordancewith one embodiment of the present invention wherein the broken lineindicates the relative positions between various elements;

FIGS. 3A and 3B are photographs showing a side by side comparison ofexemplary non-porous and porous base plates of substrates of test cardassemblies taken using a scanning electron microscope;

FIG. 4 is a flowchart schematically illustrating one embodiment of thediagnostic method for determining HCT and PT according to the presentinvention;

FIG. 5 is experimental graph showing the change of impedance vs.coagulation time in seconds, which illustrates a change in slope as awhole blood sample coagulates by a typical impedance measurement method;

FIG. 6 is experimental graph showing the change of reactance vs.coagulation time in seconds, which illustrates a change in slope as awhole blood sample coagulates by the reactance measurement method;

FIGS. 7 and 8 are the relation of HCT and impedance (FIG. 7) and HCT andreactance (FIG. 8) calculated from the experimental graphs of FIGS. 5and 6, respectively;

FIGS. 9 and 10 depict exemplary impedance and reactance values measuredby an LCR meter every 0.5 second over 60 seconds;

FIGS. 11 and 12 depict exemplary PT vs. impedance change ratecalibration curve and PT vs. reactance change rate calibration curve,respectively;

FIGS. 13 and 14 depict exemplary graphs of Calibrated PT vs. Real PT byimpedance and Calibrated PT vs. Real PT by reactance, respectively;

FIGS. 15A and 15B are experimental graphs showing the blood coagulationanalyses using a porous substrate and a non-porous substrate,respectively; and

FIGS. 16A-16C are experimental graphs showing blood coagulation analysesat different frequencies using the reactance methods of measurementaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to several exemplary embodiments ofthe invention, examples of which are illustrated in the accompanyingdrawings and photographs. Wherever possible, the same reference numbersare used in the drawings and the description to refer to the same orlike parts. In the drawings, the shape and thickness of an embodimentmay be exaggerated for clarity and convenience. This description will bedirected in particular to elements forming part of, or cooperating moredirectly with, apparatus in accordance with the present invention. It isto be understood that elements not specifically shown or described maytake various forms well known to those skilled in the art. Further, whena layer is referred to as being on another layer or “on” a substrate, itmay be directly on the other layer or on the substrate, or interveninglayers may also be presented.

Some exemplary embodiments of the present invention are described ingreater detail by referring to the drawings and photographs thataccompany the present application. It should be noted that the featuresillustrated in the drawings are not necessarily drawn to scale.Descriptions of well-known components, materials, and process techniquesmay be omitted so as to not unnecessarily obscure the embodiments of theinvention. Any devices, components, materials, and steps described inthe embodiments are only for illustration and not intended to limit thescope of the present invention.

In view of the aforementioned problems noted of the conventionaltechnologies, the following embodiments provide a system and methods fordetermining hematocrit (HCT) and prothrombin time (PT) of a sample (forexample, a blood sample) by performing a reactance analysis on thesample, also referred to as a reactance measurement module. Measurementsfor blood coagulation, or HCT, by performing a reactance analysis asdisclosed herein may be suitably used in quantitative analysis ofprothrombin time (i.e., clotting time). As used herein, the term“hematocrit” refers to the percentage of packed red blood cells in avolume of whole blood.

One exemplary embodiment of the invention provides an electrode-typesensor device with a sample test card including one or more pairs ofelectrodes. The base plate of the test card may be made of porousmaterials or non-porous materials, but preferably the substrate is madeof porous materials as described and shown herein. The electrodes may bemade of carbon or other conductive materials, but preferably are made ofprecious metals which include, for example, gold, silver, palladium,platinum, nickel, alloys thereof, and combinations thereof known tothose skilled in the art. In one aspect of the invention, an alternatingcurrent (AC) module or an AC/DC power source provides a test signal tothe blood test card with an oscillated frequency in a range betweenabout 0.1 KHz and about 50 KHz. The amplitude of voltage applied to thetest card is in a range of about 0.05 V to about 5 V. The signal isapplied to the test card to measure the reactance of the sample. As usedherein in connection with a measured quantity, the term “about” refersto that variation in the measured quantity as would be expected by theskilled artisan performing the measurement and exercising a level ofcare commensurate with the objective of the measurement and theprecision of the measuring apparatus being used.

In one preferred aspect of the invention, as blood coagulation in asample caused by enzymatic reactions proceeds, the responding signalsare received and processed by the sensor device according to the slopedifferences depending on the prothrombin time periods analyzed by thereactance measurement. In one embodiment, the electrodes may be goldelectrodes. In one example, an AC module is adopted for taking areactance measurement of the sample according to the respondingoscillated test signal sensed from the blood test card, wherein as theblood coagulation caused by the enzyme reactions proceeds the resultantsignals are received and processed by the sensor device according to theslope differences depending on the prothrombin time periods analyzed bythe reactance measurement.

Principles of performing a reactance analysis according to embodimentsof the present invention are discussed below. The impedance of an ACcircuit equals the sum of resistance (R) and the product of reactance(X) and the phase angle θ:

wherein reactance (X) is the imaginary part of the complex quantity,impedance (Z), representing the obstacles to the current flow created bya combination of inductances (L) and capacitances (C). Resistance (R) isthe real part of the complex quantity. As known to those skilled in theart, the reactance changes with changes in the frequency, changes in thecapacitance and/or changes in the inductance of the AC circuit. When thereactance of the AC circuit changes, there will be a phase changebetween the circuit's current waveform and voltage waveform. Theimpedance (Z) is defined as:Z=R+jX, and |Z|=(R ² +X ²)^(1/2)  (1)where Z is impedance, R is resistance, j is phase, and X is reactance;andX=X _(C) +X _(L) ,X _(L)=2πfL, and X _(C)=½πfC  (2)where X_(C) is capacitance reactance, X_(L) is inductance reactance, πis a ratio of the circumference of a circle to its diameter, f isfrequency, L is inductance, and C is capacitance.

In one aspect of the invention, an AC generation unit of the detectionsystem provides an AC test signal to the test card. A sample is appliedto the test card in a sample test area of the test card. As the samplebetween the electrodes is charged, the induced charges are polarized inthe electric field to form capacitances and the sample reacts somewhatlike a capacitor. As a blood sample clots, during the clotting a mediaforms between the electrodes, impeding movement of charges in the samplesuch that charges accumulate on the electrodes. The accumulated chargesthereby cause a capacitance to develop. In a preferred aspect of theinvention, since the frequency (f) of the AC generation unit of thedetection system remains constant, the inductance (L) and the inductancereactance (X_(L)) is also constant, and, therefore, the variation in thecapacitance reactance in the sample equals essentially the variation inthe overall reactance as shown by the following equation:X _(C2) −X _(C1) =X ₂ −X ₁  (3)where X_(C2)−X_(C1) is the variation in the capacitance reactance, andX₂−X₁ is the variation in the reactance.

By determining reactance variation of the sample per unit time, thecapacitance reactance variation per unit time and the capacitancevariation per unit time during characteristics of the blood clotting inthe sample can be determined. Prothrombin time (i.e., clotting time)measurement may thus be determined accurately by performing a slopecalculation with the help of the reactance measurement module of theinvention.

FIG. 1 is a schematic view of an exemplary diagnostic device formeasuring prothrombin time and/or HCT in accordance with embodiments ofthe present invention. As illustrated in FIG. 1, a diagnostic device 100for measuring prothrombin time and/or HCT of a fluid includes a relativeelectrode-type sensor device 120 and a sample test card assembly 110including one or more pairs of electrodes, wherein alternating current(AC) provided by the sensor device is used to measure and calculateprothrombin time and HCT of blood test using the reactance analysis. Inthis embodiment, the sensor device 120 includes a test card receivingunit 122 for accommodating the test card assembly 110. A temperaturemaintaining unit 134 is used for controlling and maintaining temperatureof the test card receiving unit at a constant temperature. An ACgeneration unit 124 provides an alternating current with predeterminedfrequency and voltage to the test card assembly 110. A signal retrievingunit 128 is used to retrieve a response signal from the test cardassembly. A microprocessor 130 is used for analyzing the response signaland rendering results of the HCT and/or the prothrombin time. A displayunit 136 is used for displaying inspected results of the HCT and/or theprothrombin time from the microprocessor 130.

FIG. 2 illustrates an explosive view of a sample test card for a samplein accordance with one embodiment of the present invention wherein thebroken line indicates the relative positions between various elements.The sample test card includes an insulating substrate 210, an electrodesystem 220, a separation and reaction layer 230 and a cover 240. Theinsulating substrate 210 is electrically insulating, and its materialmay include, but is not limited to, a base plate composed of porousmaterials. In preferred embodiments of the invention, the base plate ofthe test card assembly includes pores, cavities, voids or holes withdiameters in a range of about 0.1 μm to about 10 μm, about 0.01 μm toabout 100 μm, about 0.1 μm to about 50 μm, about 0.1 μm to about 20 μm,about 0.1 μm to about 5 μm, or about 5 μm to about 10 μm. The electrodesystem 220 may be made with any conductive materials, including but notlimited to carbon, gold, silver, copper, carbon silver, palladium,nickel, and other similar materials and combinations thereof accordingto the invention. The electrode system 220 includes one or more pairs ofthe noble metal electrodes, set on the same plane or on different planesrespectively. For example, a set of testing electrodes 225 includes apair of electrodes 226 and 228. According to the principles of theinvention, the electrode structure is not limited to specificarrangements of the set of testing electrodes 225 as shown or the exactnumber of electrodes as shown. Additional electrodes may be providedaccording to different application needs. The electrode system furtherelectrically connects the electrode system with a measurement device(not shown).

The separation layer 230 is depicted as including spacers 232 disposedover the electrode system 220. The separation layer 230 further mayinclude a reaction region 224 to expose a part of the reagent (notshown) and a sampling region 222. A channel 236 may connect the samplingregion 222 and reaction region 224. The size of the reaction region 224is preferably sufficient to expose part of the electrodes 226 and 228.In this example, the reaction region 224 is used for measurement of theprothrombin time, and the sampling region 222 may be used formeasurement of the HCT.

The cover 240 is disposed on the separation layer 230. In oneembodiment, the cover 240 includes an inlet 242 and a gas vent 244,which are respectively connected to the sampling region 222 and thereacting region 224. The size of the sampling space depends upon thethicknesses of the separation layer 230.

FIGS. 3A and 3B are photographs showing a side by side comparison ofexemplary non-porous and porous base plates of test card assembliestaken using a scanning electron microscope. In FIG. 3B, the diameter ofpore size of the base plate is in a range of about 0.1 μm to about 10 μmwith an average diameter of about 3.39 μm. Pore distribution on the baseplate is about 5.04×10⁶ holes/cm².

FIG. 4 is a flowchart schematically illustrating one embodiment of adiagnostic method for determining HCT and prothrombin time according tothe present invention. The method for measuring HCT and prothrombin timein FIG. 4 includes at least these steps: providing a test card assemblyto a test card receiving unit (S410); controlling and maintaining thetemperature of the test card receiving unit at a constant temperature(S420); providing a sample to be inspected to the test card assembly(S430); providing an alternating current with predetermined frequencyand voltage to the test card assembly by an AC generation unit (S430);sensing a response signal from the test card assembly and determining achange in the phase of the signals (i.e., phase shift) and calculatingthe reactance and the HCT using a microprocessor (S450); correcting theprothrombin time by reference of the HCT (S460); correcting theprothrombin time with an international normalized ratio (S470); andproviding inspected results to a display unit (S480).

According to one embodiment of the present invention, the responsesignal retrieved from the test card is digitized and converted by thediscrete fourier transform (DFT) by a microprocessor. Thus, as known tothose skilled in the art, the real value and imaginary value may bedetermined by the method shown below:

$\begin{matrix}{{{X(k)} = {{{DFT}\left\lbrack {x(n)} \right\rbrack} = {\sum\limits_{n = 0}^{N - 1}\;{{x(n)}W_{N}^{kn}}}}},{0 \leq k \leq {N - 1}},} & (1) \\{W_{N} = {\mathbb{e}}^{{- j}\frac{2\pi}{N}}} & (2)\end{matrix}$where X(k) is fourier value of digital signal, x(n) is original value ofdigital signal, n is current point of digital signal, and N is totalnumber of digital signal. Further, the phase may be calculated by theimaginary value and real value according to the following formula:Phase=tan⁻¹(Im/Re)  (3)where Im is the imaginary value (i.e., due to the reactance) and Re isthe real value (i.e., due to the real resistance). As shown in theformula (3), the phase will shift by the change in the reactance in thesample.

In some embodiments, the step of determining phase change in S450 (i.e.,phase change due to reactance) as discussed above includes the steps of:calculating the magnitude of impedance from the measured response signaland the applied voltage; calculating a change in the phase; calculatingreactance from the change in the phase. As will be appreciated, sincethe alternating current has a constant frequency, the change in thephase angle is primarily due to the change in capacitance in the sampleas described above. Since any changes in capacitance in the samplecreates a change in the reactance portion of impedance, the change inreactance may be used to yield accurate HCT and PT values as discussedbelow and shown on the attached Figures.

From the measured reactance the HCT can be calculated by interpolation.One example of calculating the HCT is described below. A traditionalmethod of using the impedance in calculating the HCT is also describedas a comparison.

FIGS. 5 and 6 depict experimental graphs showing the impedance andreactance increase with higher percentage HCT, respectively. As shown inthe graph in FIG. 6, in this particular example, in the 11th second, thereactance of different HCT (29, 39 and 47%) was respectively 620.29,652.17 and 676.59 ohm. Then, the relation of HCT and impedance (FIG. 7)or HCT and reactance (FIG. 8) were calculated. As shown in FIG. 5, inthis particular example, the optimal impedance sampling time of HCT wasat about 20 seconds or more, and Impedance vs. HCT calibration curve hasan equation of y=90.253x+2347.7. Further, as shown in FIG. 6, theoptimal reactance sampling time of HCT was at about 11 seconds or more,and Reactance vs. HCT calibration curve had an equation ofy=3.1304x+529.68. As will be appreciated, x is the HCT, and y is theimpedance or reactance, respectively.

FIGS. 9 and 10 depict exemplary impedance and reactance values measuredby an LCR meter every 0.5 second over 60 seconds. In one example, wholeblood was collected from a subject, and various samples were prepared byadding different amounts of anticoagulant drug (heparin) to thecollected whole blood. In one instance, the concentration of heparinused to modulate the coagulation time (PT) was between about 1 U andabout 30 U per milliliter. Then, the blood samples with different PTwere analyzed to measure the impedance or reactance by a LCR meter(Hioki Model No. 3532-50).

FIGS. 11 and 12 depict an exemplary PT vs. impedance change ratecalibration curve and an exemplary PT vs. reactance change ratecalibration curve, respectively. In this particular example, theimpedance or reactance change rate was computed every 10 sec by the LCRmeter. For example, the change rate from 30 second to 40 second wascomputed by following formula: Impedance change rate 30 to40=(Z₄₀−Z₃₀)/(Time₄₀−Time₃₀), wherein Z is impedance, and Reactancechange rate 30 to 40=(X₄₀−X₃₀)/(Time₄₀−Time₃₀), wherein X is reactance.The computation step was repeated to compute the impedance or reactancechange rate of the blood for samples with different PTs, and therebydetermine the PT vs. impedance change rate calibration curve (FIG. 7)and the PT vs. reactance change rate calibration curve (FIG. 8). The PTvs. impedance change rate calibration curve had an equation ofy=−0.1849x+4.562, and the PT vs. reactance change rate calibration curvehad an equation of y=−0.0256x+0.3604. As will be appreciated, x is thereal PT, and y is the impedance or reactance change rate.

As shown in FIGS. 11 and 12, the diagnostic results of the reactancemeasurement method (FIG. 12) showed a superior standard deviation (SDvalue) compared to the results from the typical impedance measurementmethod (FIG. 11). Specifically, adoption of the reactance measurementaccording to the present invention is advantageous compared to thetypical impedance measurement method in that the standard deviation inthe reactance measurement is significantly reduced and the linearregression value (R2) in this example is about 0.9986. Accordingly, thereactance measurement method according to the present invention yields amore accurate result than the traditional impedance measurement method,and the slope deviation is acceptable even as blood coagulation processis extended, thus easily adjusting the values.

On the other hand, in this particular example, the linear regression(R²) for the PT vs. impedance change rate calibration curve was about0.939 as shown in FIG. 11. Accordingly, the impedance method is morelikely to cause an inaccurate measurement.

In the above example, the impedance and reactance of a blood sample weremeasured by the LCR meter, and its impedance change rate and reactancechange rate were calculated. Specifically, the HCT was calculated byusing the HCT calibration curve with the formula,HCT=(impedance−2347.7)/90.253 or HCT=(reactance−529.68)/3.1304. Then,the real PT is calculated by the PT calibration curve. Different HCT maycorrespond with different PT calibration curve. In this particularexample, the PT was calculated by using the PT calibration curve withthe formula, PT=(impedance change rate−4.562)/−0.1849 or PT=(reactancechange rate−0.3604)/−0.0256.

FIGS. 13 and 14 depict exemplary graphs of Calibrated PT vs. Real PT byimpedance and Calibrated PT vs. Real PT by reactance, respectively. ThePT values computed by the calibration curve and the real PT valuesmeasured by the automated blood coagulation analyzer (Sysmex CA-500series) were compared.

The increase or decrease in the HCT influences blood clotting time (PT)and the impendence or reactance change rate value. Specifically, ahigher HCT causes the impendence or reactance change rate value toincrease. Hence, the PT determination process in some embodiments mayinclude an HCT correction step. A device according to some embodimentsof the present invention includes an internal memory for storingexperimental results of PT values for different HCT samples that can beused to correct PT by reference of the HCT. For example, a data base maybe built up in the meter that includes serial experiment results of realPT values for different HCT samples. For example, values between 30 HCT% to 60 HCT % may be stored for every 5 HCT % of real PT value.Therefore, for example, when PT value for 35% HCT (PT₁) and PT value for40% HCT (PT₂) are stored in the memory, a PT₃ value for 38% HCT can beestimated to be (PT₁×⅖)+(PT₂×⅗).

In one aspect of the invention, the PT values may be corrected with aninternational normalized ratio shown in the following formula:

$\begin{matrix}{{INR} = \left( \frac{{Patient}\mspace{14mu}{PT}}{{Mean}\mspace{14mu}{PT}} \right)^{ISI}} & (4)\end{matrix}$where INR is international normalized ratio, PT is prothrombin time, andISI is an international sensitivity index.

FIGS. 15A and 15B are experimental graphs showing the blood coagulationanalyses using a porous substrate and a non-porous substrate for thetest card, respectively. As illustrated in the Figures, the analysesemploying a porous substrate provided superior results.

FIGS. 16A-16C depict experimental graphs showing the blood coagulationanalyses at different frequencies by the reactance measurement accordingto the present invention. Coagulation times between 15-50 seconds ismeasured for the blood samples at frequencies of 0.1 kHz, 10 kHz and 50kHz, and test results R² were obtained by calculating regressionanalysis of 0.9636, 0.9923, and 0.9858 respectively. In this case, theregression analysis indicates that using frequencies of 10 KHz and 50KHz provide greater accuracy as compared to a frequency of 0.1 KHz.

While the invention has been described by way of examples and in termsof preferred embodiments, it would be apparent to those skilled in theart to make various equivalent replacements, amendments andmodifications in view of specification of the invention. Therefore, thescope of the appended claims should be accorded the broadestinterpretation so as to encompass all such replacements, amendments andmodifications without departing from the spirit and scope of theinvention.

The invention claimed is:
 1. A diagnostic device for measuringhematocrit (HCT) or prothrombin time (PT) of a sample, comprising: asensor device; and a test card assembly including one or more pairs ofelectrodes, wherein the sensor device comprises a test card receivingunit for accommodating the test card assembly; a power source forproviding an AC test signal at a constant frequency to electrodes of thetest card assembly; a signal retrieving unit coupled to the test cardassembly; a microprocessor in the sensor device programmed for (i)performing a reactance analysis of a sample provided on the test cardassembly by comparing the AC test signal and a response signal from thetest card assembly in response to the AC test signal, calculating achange in phase of the AC test signal, and calculating a reactance fromthe change in phase of the AC test signal, and (ii) determining PT orHCT of the sample; and, a display for displaying the PT or HCT of thesample.
 2. The diagnostic device as claimed in claim 1, wherein thereactance analysis further comprises: calculating a reactance due to achange in capacitance, and transforming the change in capacitance withalgorithms and correcting to the prothrombin time by reference of theHCT.
 3. The diagnostic device as claimed in claim 1, the reactanceanalysis further comprises correcting the PT with an internationalnormalized ratio.
 4. The diagnostic device as claimed in claim 1,wherein the test card assembly comprises one or more pairs of the noblemetal electrodes, set on the same plane.
 5. The diagnostic device asclaimed in claim 1, wherein the test card assembly comprises one or morepairs of the electrodes, set on different planes.
 6. The diagnosticdevice as claimed in claim 1, wherein the test card assembly comprises abase plate comprising a porous material that includes holes, voids orcavities thereon.
 7. The diagnostic device as claimed in claim 6,wherein the base plate of the test card assembly comprises pores such asholes, voids or cavities thereon, with diameters approximately in arange of about 0.1 μm to about 10 μm.
 8. The diagnostic device asclaimed in claim 7, wherein the microprocessor further transforms thecapacitance with algorithms, corrects to the PT by reference of the HCT;and calculates the PT with an international normalized ratio.